Ultra bright dimeric or polymeric dyes

ABSTRACT

Compounds useful as fluorescent or colored dyes are disclosed. The compounds have the following structure (I): 
                         
including stereoisomers, salts and tautomers thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , L 3 , L 4 , M, m and n are as defined herein. Methods associated with preparation and use of such compounds are also provided.

BACKGROUND Field

The present invention is generally directed to dimeric and polymericfluorescent or colored dyes, and methods for their preparation and usein various analytical methods.

Description of the Related Art

Fluorescent and/or colored dyes are known to be particularly suitablefor applications in which a highly sensitive detection reagent isdesirable. Dyes that are able to preferentially label a specificingredient or component in a sample enable the researcher to determinethe presence, quantity and/or location of that specific ingredient orcomponent. In addition, specific systems can be monitored with respectto their spatial and temporal distribution in diverse environments.

Fluorescence and colorimetric methods are extremely widespread inchemistry and biology. These methods give useful information on thepresence, structure, distance, orientation, complexation and/or locationfor biomolecules. In addition, time-resolved methods are increasinglyused in measurements of dynamics and kinetics. As a result, manystrategies for fluorescence or color labeling of biomolecules, such asnucleic acids and protein, have been developed. Since analysis ofbiomolecules typically occurs in an aqueous environment, the focus hasbeen on development and use of water soluble dyes.

Highly fluorescent or colored dyes (“brighter”) are desirable since useof such dyes increases the signal to noise ratio and provides otherrelated benefits. Accordingly, attempts have been made to increase thesignal from known fluorescent and/or colored moieties. For example,dimeric and polymeric compounds comprising two or more fluorescentand/or colored moieties have been prepared in anticipation that suchcompounds would result in brighter dyes. However, as a result ofintramolecular fluorescence quenching, the dimeric and polymeric dyesdid not achieve the desired increase in brightness.

There is thus a need in the art for water soluble dyes having anincreased molar brightness. Ideally, such dyes and biomarkers should beintensely colored or fluorescent and should be available in a variety ofcolors and fluorescent wavelengths. The present invention fulfills thisneed and provides further related advantages.

BRIEF SUMMARY

In brief, the present invention is generally directed to compoundsuseful as water soluble, fluorescent or colored dyes and probes thatenable visual detection of analyte molecules, such as biomolecules, aswell as reagents for their preparation. Methods for visually detectingan analyte molecule using the dyes are also described.

Embodiments of the presently disclosed dyes include two or morefluorescent and/or colored moieties covalently linked by a linker whichis typically charged at pH's ranging from 1-14, for example at pH'sabove 7. In contrast to previous reports of dimeric and/or polymericdyes, the present dyes are significantly brighter than the correspondingmonomeric dye compound. While, not wishing to be bound by theory, it isbelieved that electrostatic repulsion of the charged linker acts tomaintain the spatial distant between the fluorescent and/or coloredmoieties, and thus intramolecular fluorescence quenching is reducedand/or eliminated.

The water soluble, fluorescent or colored dyes of the invention areintensely colored and/or fluorescent and can be readily observed byvisual inspection or other means. In some embodiments the compounds maybe observed without prior illumination or chemical or enzymaticactivation. By appropriate selection of the dye, as described herein,visually detectable analyte molecules of a variety of colors may beobtained.

In one embodiment, compounds having the following structure (I) areprovided:

or a stereoisomer, tautomer or salt thereof, wherein R¹, R², R³, R⁴, R⁵,L¹, L², L³, L⁴, M, m and n are as defined herein.

In another embodiment, a method for staining a sample is provided, themethod comprises adding to said sample a representative compound asdescribed herein in an amount sufficient to produce an optical responsewhen said sample is illuminated at an appropriate wavelength.

In still other embodiments, the present disclosure provides a method forvisually detecting an analyte molecule, comprising:

-   -   (a) providing a representative compound described herein; and    -   (b) detecting the compound by its visible properties.

Other disclosed methods include a method for visually detecting abiomolecule, the method comprising:

-   -   (a) admixing any of the disclosed compounds with one or more        biomolecules; and    -   (b) detecting the compound by its visible properties.

Other embodiments are directed to a composition comprising any one ofthe disclosed compounds and one or more biomolecules. Use of suchcomposition in analytical methods for detection of the one or morebiomolecules is also provided.

These and other aspects of the invention will be apparent upon referenceto the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, identical reference numbers identify similar elements.The sizes and relative positions of elements in the figures are notnecessarily drawn to scale and some of these elements are arbitrarilyenlarged and positioned to improve figure legibility. Further, theparticular shapes of the elements as drawn are not intended to conveyany information regarding the actual shape of the particular elements,and have been solely selected for ease of recognition in the figures.

FIG. 1 provides UV absorbance spectra for comparative dye compounds.

FIG. 2 is an overlay of fluorescent emission spectra for comparative dyecompounds.

FIG. 3 presents UV absorption spectra for exemplary dye compounds.

FIG. 4 shows fluorescent emission spectra for the dye compounds of FIG.3.

FIG. 5 is another overlay of UV absorption spectra of representative dyecompounds.

FIG. 6 illustrates fluorescent emission spectra for representative dyecompounds.

FIG. 7 provides another overlay of fluorescent emission spectra forrepresentative dye compounds relative to the parent fluorophore.

FIG. 8 is another overlay of fluorescent emission spectra forrepresentative dye compounds relative to the parent fluorophore.

FIG. 9 provides additional fluorescence emission spectra ofrepresentative compounds relative to the parent fluorophore.

FIGS. 10a and 10b provides dose dependent data from a spike and recoveryassay of cells.

FIG. 11 provides flow cytometry data correlating live/dead gating of dyestained cells using DNA intercalated 7-AAD.

FIG. 12 provides flow cytometry data showing a vitality gradient thatcorrelates to graded states of membrane permeability.

FIG. 13 illustrates sensitive detection of vitality states between twodifferent cultures and morphology interpretations.

FIGS. 14a and 14b illustrate patterns of mitosis found in dead cells.

FIGS. 15a and 15b illustrate patterns of proliferative effects of deadand apoptotic cells.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of theinvention. However, one skilled in the art will understand that theinvention may be practiced without these details.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is, as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

“Amino” refers to the —NH₂ group.

“Carboxy” refers to the —CO₂H group.

“Cyano” refers to the —CN group.

“Formyl” refers to the —C(═O)H group.

“Hydroxy” or “hydroxyl” refers to the —OH group.

“Imino” refers to the ═NH group.

“Nitro” refers to the —NO₂ group.

“Oxo” refers to the ═O substituent group.

“Sulfhydryl” refers to the —SH group.

“Thioxo” refers to the ═S group.

“Alkyl” refers to a straight or branched hydrocarbon chain groupconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds),having from one to twelve carbon atoms (C₁-C₁₂ alkyl), preferably one toeight carbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆alkyl), and which is attached to the rest of the molecule by a singlebond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl),n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl,2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl,penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and thelike. An “alkenyl” is an alkyl comprising at least one carbon-carbondouble bond. An “alkynyl” is an alkyl comprising at least onecarbon-carbon triple bond. Unless stated otherwise specifically in thespecification, alkyl, alkenyl and alkynyl groups are optionallysubstituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a substituentgroup, consisting solely of carbon and hydrogen, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds), andhaving from one to twelve carbon atoms, e.g., methylene, ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. An “alkenylene” is an alkylenecomprising at least one carbon-carbon double bond. An “alkynylene” is analkylene comprising at least one carbon-carbon triple bond. The alkylenechain is attached to the rest of the molecule through a single or doublebond and to the substituent group through a single or double bond. Thepoints of attachment of the alkylene chain to the rest of the moleculeand to the substituent group can be through one carbon or any twocarbons within the chain. Unless stated otherwise specifically in thespecification, alkylene, alkenylene and alkynylene are optionallysubstituted.

“Alkylether” refers to any alkyl group as defined above, wherein atleast one carbon-carbon bond is replaced with a carbon-oxygen bond. Thecarbon-oxygen bond may be on the terminal end (as in an alkoxy group) orthe carbon oxygen bond may be internal (i.e., C—O—C). Alkylethersinclude at least one carbon oxygen bond, but may include more than one.For example, polyethylene glycol (PEG) is included within the meaning ofalkylether. Unless stated otherwise specifically in the specification,an alkylether group is optionally substituted. For example, in someembodiments and alkylether is substituted with an alcohol or phosphate.

“Alkoxy” refers to a group of the formula —OR_(a) where R_(a) is analkyl group as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, an alkoxygroup is optionally substituted.

“Heteroalkylene” refers to an alkylene group comprising at least oneheteroatom (e.g., N, O or S). In some embodiments, the heteroatom iswithin the alkylene chain (i.e., the heteroalkylene comprises at leastone carbon-heteroatom-carbon bond. In other embodiments, the heteroatomis at a terminus of the alkylene and thus serves to join the alkylene tothe remainder of the molecule (e.g., M1-H-A-M2, where M1 and M2 areportions of the molecule, H is a heteroatom and A is an alkylene).“Heteroalkenylene” is a heteroalkylene comprising at least onecarbon-carbon double bond. “Heteroalkynylene” is a heteroalkylenecomprising at least one carbon-carbon triple bond. Unless statedotherwise specifically in the specification, heteroalkylene,heteroalkenylene and heteroalkynylene are optionally substituted.

An exemplary heteroalkylene linking group is illustrated below:

Multimers of the above C-linker are included in various embodiments ofheteroalkylene linkers.

“Heteroatomic” in reference to a “heteroatomic linker” refers to alinker group consisting of one or more heteroatom. Exemplaryheteroatomic linkers include single atoms selected from the groupconsisting of O, N, P and S, and multiple heteroatoms for example alinker having the formula —P(O⁻)(═O)O— or —OP(O⁻)(═O)O— and multimersand combinations thereof.

“Phosphate” refers to the —OP(═O)(R_(a))R_(b) group, wherein R_(a) isOH, O or OR_(E); and R_(b) is OH, O⁻, OR_(E), a thiophosphate group or afurther phosphate group, wherein R_(c) is a counter ion (e.g., Na+ andthe like).

“Phosphoalkyl” refers to the —OP(═O)(R_(a))R_(b) group, wherein R_(a) isOH, O⁻ or OR_(c) ⁻; and R_(b) is —Oalkyl, wherein R_(c) is a counter ion(e.g., Na+ and the like). Unless stated otherwise specifically in thespecification, a phosphoalkyl group is optionally substituted. Forexample, in certain embodiments, the —Oalkyl moiety in a phosphoalkylgroup is optionally substituted with one or more of hydroxyl, amino,sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether.

“Phosphoalkylether” refers to the —OP(═O)(R_(a))R_(b) group, whereinR_(a) is OH, O⁻ or OR_(c); and R_(b) is —Oalkylether, wherein R_(c) is acounter ion (e.g., Na+ and the like). Unless stated otherwisespecifically in the specification, a phosphoalkylether group isoptionally substituted. For example, in certain embodiments, the—Oalkylether moiety in a phosphoalkylether group is optionallysubstituted with one or more of hydroxyl, amino, sulfhydryl, phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether.

“Thiophosphate” refers to the —OP(═R_(a))(R_(b))R_(c) group, whereinR_(a) is O or S; R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); and R_(c) is OH,SH, O⁻, S⁻, OR_(d), SR_(d), a phosphate group or a further thiophosphategroup, wherein R_(d) is a counter ion (e.g., Na+ and the like) andprovided that: i) R_(a) is S; ii) R_(b) is S⁻ or SR_(d) ⁻; iii)R_(c) isSH, S⁻ or SR_(d); or iv) a combination of i), ii) and/or iii).

“Thiophosphoalkyl” refers to the —OP(═R_(a))(R_(b))R_(c) group, whereinR_(a) is O or S; R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d) ⁻; and R_(c) is—Oalkyl, wherein R_(d) is a counter ion (e.g., Na+ and the like) andprovided that: R_(a) is S or R_(b) is S⁻ or SR_(d); or provided thatR_(a) is S and R_(b) is S⁻ or SR_(d). Unless stated otherwisespecifically in the specification, a thiophosphoalkyl group isoptionally substituted. For example, in certain embodiments, the —Oalkylmoiety in a thiophosphoalkyl group is optionally substituted with one ormore of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate,phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether.

“Thiophosphoalkylether” refers to the —OP(═R_(a))(R_(b))R_(c) group,wherein R_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); andR_(c) is —Oalkylether, wherein R_(d) is a counter ion (e.g., Na+ and thelike) and provided that: R_(a) is S or R_(b) is S⁻ or SR_(d); orprovided that R_(a) is S and R_(b) is S⁻ or SR_(d). Unless statedotherwise specifically in the specification, a thiophosphoalkylethergroup is optionally substituted. For example, in certain embodiments,the —Oalkylether moiety in a thiophosphoalkyl group is optionallysubstituted with one or more of hydroxyl, amino, sulfhydryl, phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether.

“Carbocyclic” refers to a stable 3- to 18-membered aromatic ornon-aromatic ring comprising 3 to 18 carbon atoms. Unless statedotherwise specifically in the specification, a carbocyclic ring may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems, and may be partially or fullysaturated. Non-aromatic carbocyclyl radicals include cycloalkyl, whilearomatic carbocyclyl radicals include aryl. Unless stated otherwisespecifically in the specification, a carbocyclic group is optionallysubstituted.

“Cycloalkyl” refers to a stable non-aromatic monocyclic or polycycliccarbocyclic ring, which may include fused or bridged ring systems,having from three to fifteen carbon atoms, preferably having from threeto ten carbon atoms, and which is saturated or unsaturated and attachedto the rest of the molecule by a single bond. Monocyclic cyclocalkylsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptly, and cyclooctyl. Polycyclic cycloalkyls include, forexample, adamantyl, norbornyl, decalinyl,7,7-dimethyl-bicyclo-[2.2.1]heptanyl, and the like. Unless statedotherwise specifically in the specification, a cycloalkyl group isoptionally substituted.

“Aryl” refers to a ring system comprising at least one carbocyclicaromatic ring. In some embodiments, an aryl comprises from 6 to 18carbon atoms. The aryl ring may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems. Aryls include, but are not limited to, aryls derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene,indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene,and triphenylene. Unless stated otherwise specifically in thespecification, an aryl group is optionally substituted.

“Heterocyclic” refers to a stable 3- to 18-membered aromatic ornon-aromatic ring comprising one to twelve carbon atoms and from one tosix heteroatoms selected from the group consisting of nitrogen, oxygenand sulfur. Unless stated otherwise specifically in the specification,the heterocyclic ring may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heterocyclicring may be optionally oxidized; the nitrogen atom may be optionallyquaternized; and the heterocyclic ring may be partially or fullysaturated. Examples of aromatic heterocyclic rings are listed below inthe definition of heteroaryls (i.e., heteroaryl being a subset ofheterocyclic). Examples of non-aromatic heterocyclic rings include, butare not limited to, dioxolanyl, thienyl[1,3]dithianyl,decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl,isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl,piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl,pyrazolopyrimidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl,trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiomorpholinyl,thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl.Unless stated otherwise specifically in the specification, aheterocyclic group is optionally substituted.

“Heteroaryl” refers to a 5- to 14-membered ring system comprising one tothirteen carbon atoms, one to six heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur, and at least one aromaticring. For purposes of certain embodiments of this invention, theheteroaryl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heteroarylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized. Examples include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzthiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,benzoxazolinonyl, benzimidazolthionyl, carbazolyl, cinnolinyl,dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl,imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl,isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl,oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl,1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl,1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl,phthalazinyl, pteridinyl, pteridinonyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyridinonyl, pyrazinyl, pyrimidinyl, pryrimidinonyl,pyridazinyl, pyrrolyl, pyrido[2,3-d]pyrimidinonyl, quinazolinyl,quinazolinonyl, quinoxalinyl, quinoxalinonyl, quinolinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl,thieno[3,2-d]pyrimidin-4-onyl, thieno[2,3-d]pyrimidin-4-onyl, triazolyl,tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl). Unless statedotherwise specifically in the specification, a heteroaryl group isoptionally substituted.

“Fused” refers to a ring system comprising at least two rings, whereinthe two rings share at least one common ring atom, for example twocommon ring atoms. When the fused ring is a heterocyclyl ring or aheteroaryl ring, the common ring atom(s) may be carbon or nitrogen.Fused rings include bicyclic, tricyclic, tertracyclic, and the like.

The term “substituted” used herein means any of the above groups (e.g.,alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene,heteroalkylene, heteroalkenylene, heteroalkynylene, alkoxy, alkylether,phosphate, phosphoalkyl, phosphoalkylether, thiophosphate,thiophosphoalkyl, thiophosphoalkylether, carbocyclic, cycloalkyl, aryl,heterocyclic and/or heteroaryl) wherein at least one hydrogen atom(e.g., 1, 2, 3 or all hydrogen atoms) is replaced by a bond to anon-hydrogen atom such as, but not limited to: a halogen atom such as F,Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxygroups, and ester groups; a sulfur atom in groups such as thiol groups,thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups;a nitrogen atom in groups such as amines, amides, alkylamines,dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides,imides, and enamines; a silicon atom in groups such as trialkylsilylgroups, dialkylarylsilyl groups, alkyldiarylsilyl groups, andtriarylsilyl groups; and other heteroatoms in various other groups.“Substituted” also means any of the above groups in which one or morehydrogen atoms are replaced by a higher-order bond (e.g., a double- ortriple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl,and ester groups; and nitrogen in groups such as imines, oximes,hydrazones, and nitriles. For example, “substituted” includes any of theabove groups in which one or more hydrogen atoms are replaced with—NR_(g)R_(h), —NR_(g)C(═O)R_(h), —NR_(g)C(═O)NR_(g)R_(h),—NR_(g)C(═O)OR_(h), —NR_(g)SO₂R_(h), —OC(═O)NR_(g)R_(h), —OR_(g),—SR_(g), —SOR_(g), —SO₂R_(g), —OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and—SO₂NR_(g)R_(h). “Substituted also means any of the above groups inwhich one or more hydrogen atoms are replaced with —C(═O)R_(g),—C(═O)OR_(g), —C(═O)NR_(g)R_(h), —CH₂SO₂R_(g), —CH₂SO₂NR_(g)R_(h). Inthe foregoing, R_(g) and R_(h) are the same or different andindependently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl,N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/orheteroarylalkyl. “Substituted” further means any of the above groups inwhich one or more hydrogen atoms are replaced by a bond to an amino,cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy,alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl,N-heteroaryl and/or heteroarylalkyl group. In addition, each of theforegoing substituents may also be optionally substituted with one ormore of the above substituents.

“Conjugation” refers to the overlap of one p-orbital with anotherp-orbital across an intervening sigma bond. Conjugation may occur incyclic or acyclic compounds. A “degree of conjugation” refers to theoverlap of at least one p-orbital with another p-orbital across anintervening double bond. For example, 1,3-butadine has one degree ofconjugation, while benzene and other aromatic compounds typically havemultiple degrees of conjugation. Fluorescent and colored compoundstypically comprise at least one degree of conjugation.

“Fluorescent” refers to a molecule which is capable of absorbing lightof a particular frequency and emitting light of a different frequency.Fluorescence is well-known to those of ordinary skill in the art.

“Colored” refers to a molecule which absorbs light within the coloredspectrum (i.e., red, yellow, blue and the like).

A “linker” refers to a contiguous chain of at least one atom, such ascarbon, oxygen, nitrogen, sulfur, phosphorous and combinations thereof,which connects a portion of a molecule to another portion of the samemolecule or to a different molecule, moiety or solid support (e.g.,microparticle). Linkers may connect the molecule via a covalent bond orother means, such as ionic or hydrogen bond interactions.

The term “biomolecule” refers to any of a variety of biologicalmaterials, including nucleic acids, carbohydrates, amino acids,polypeptides, glycoproteins, hormones, aptamers and mixtures thereof.More specifically, the term is intended to include, without limitation,RNA, DNA, oligonucleotides, modified or derivatized nucleotides,enzymes, receptors, prions, receptor ligands (including hormones),antibodies, antigens, and toxins, as well as bacteria, viruses, bloodcells, and tissue cells. The visually detectable biomolecules of theinvention (i.e., compounds of structure (I) having a biomolecule linkedthereto) are prepared, as further described herein, by contacting abiomolecule with a compound having a reactive group that enablesattachment of the biomolecule to the compound via any available atom orfunctional group, such as an amino, hydroxy, carboxyl, or sulfhydrylgroup on the biomolecule.

The terms “visible” and “visually detectable” are used herein to referto substances that are observable by visual inspection, without priorillumination, or chemical or enzymatic activation. Such visuallydetectable substances absorb and emit light in a region of the spectrumranging from about 300 to about 900 nm. Preferably, such substances areintensely colored, preferably having a molar extinction coefficient ofat least about 40,000, more preferably at least about 50,000, still morepreferably at least about 60,000, yet still more preferably at leastabout 70,000, and most preferably at least about 80,000 M⁻¹ cm⁻¹. Thecompounds of the invention may be detected by observation with the nakedeye, or with the aid of an optically based detection device, including,without limitation, absorption spectrophotometers, transmission lightmicroscopes, digital cameras and scanners. Visually detectablesubstances are not limited to those which emit and/or absorb light inthe visible spectrum. Substances which emit and/or absorb light in theultraviolet (UV) region (about 10 nm to about 400 nm), infrared (IR)region (about 700 nm to about 1 mm), and substances emitting and/orabsorbing in other regions of the electromagnetic spectrum are alsoincluded with the scope of “visually detectable” substances.

For purposes of the invention, the term “photostable visible dye” refersto a chemical moiety that is visually detectable, as definedhereinabove, and is not significantly altered or decomposed uponexposure to light. Preferably, the photostable visible dye does notexhibit significant bleaching or decomposition after being exposed tolight for at least one hour. More preferably, the visible dye is stableafter exposure to light for at least 12 hours, still more preferably atleast 24 hours, still yet more preferably at least one week, and mostpreferably at least one month. Nonlimiting examples of photostablevisible dyes suitable for use in the compounds and methods of theinvention include azo dyes, thioindigo dyes, quinacridone pigments,dioxazine, phthalocyanine, perinone, diketopyrrolopyrrole,quinophthalone, and truarycarbonium.

As used herein, the term “perylene derivative” is intended to includeany substituted perylene that is visually detectable. However, the termis not intended to include perylene itself. The terms “anthracenederivative”, “naphthalene derivative”, and “pyrene derivative” are usedanalogously. In some preferred embodiments, a derivative (e.g.,perylene, pyrene, anthracene or naphthalene derivative) is an imide,bisimide or hydrazamimide derivative of perylene, anthracene,naphthalene, or pyrene.

The visually detectable molecules of the invention are useful for a widevariety of analytical applications, such as biochemical and biomedicalapplications, in which there is a need to determine the presence,location, or quantity of a particular analyte (e.g., biomolecule). Inanother aspect, therefore, the invention provides a method for visuallydetecting a biomolecule, comprising: (a) providing a biological systemwith a visually detectable biomolecule comprising the compound ofstructure (I) linked to a biomolecule; and (b) detecting the biomoleculeby its visible properties. For purposes of the invention, the phrase“detecting the biomolecule by its visible properties” means that thebiomolecule, without illumination or chemical or enzymatic activation,is observed with the naked eye, or with the aid of a optically baseddetection device, including, without limitation, absorptionspectrophotometers, transmission light microscopes, digital cameras andscanners. A densitometer may be used to quantify the amount of visuallydetectable biomolecule present. For example, the relative quantity ofthe biomolecule in two samples can be determined by measuring relativeoptical density. If the stoichiometry of dye molecules per biomoleculeis known, and the extinction coefficient of the dye molecule is known,then the absolute concentration of the biomolecule can also bedetermined from a measurement of optical density. As used herein, theterm “biological system” is used to refer to any solution or mixturecomprising one or more biomolecules in addition to the visuallydetectable biomolecule. Nonlimiting examples of such biological systemsinclude cells, cell extracts, tissue samples, electrophoretic gels,assay mixtures, and hybridization reaction mixtures.

“Solid support” refers to any solid substrate known in the art forsolid-phase support of molecules, for example a “microparticle” refersto any of a number of small particles useful for attachment to compoundsof the invention, including, but not limited to, glass beads, magneticbeads, polymeric beads, nonpolymeric beads, and the like. In certainembodiments, a microparticle comprises polystyrene beads.

“Base pairing moiety” refers to a heterocyclic moiety capable ofhybridizing with a complementary heterocyclic moiety via hydrogen bonds(e.g., Watson-Crick base pairing). Base pairing moieties include naturaland unnatural bases. Non-limiting examples of base pairing moieties areRNA and DNA bases such adenosine, guanosine, thymidine, cytosine anduridine and analogues thereof.

Embodiments of the invention disclosed herein are also meant toencompass all compounds of structure (I) being isotopically-labelled byhaving one or more atoms replaced by an atom having a different atomicmass or mass number. Examples of isotopes that can be incorporated intothe disclosed compounds include isotopes of hydrogen, carbon, nitrogen,oxygen, phosphorous, fluorine, chlorine, and iodine, such as ²H, ³H,¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³Iand ¹²⁵I, respectively.

Isotopically-labeled compounds of structure (I) can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described below and in the followingExamples using an appropriate isotopically-labeled reagent in place ofthe non-labeled reagent previously employed.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Optional” or “optionally” means that the subsequently described eventor circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted alkyl” means that thealkyl group may or may not be substituted and that the descriptionincludes both substituted alkyl groups and alkyl groups having nosubstitution.

“Salt” includes both acid and base addition salts.

“Acid addition salt” refers to those salts which are formed withinorganic acids such as, but not limited to, hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and organic acids such as, but not limited to, acetic acid,2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid,aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoicacid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproicacid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid,galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid,glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid,glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid,lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid,malonic acid, mandelic acid, methanesulfonic acid, mucic acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid,1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid,oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamicacid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid,stearic acid, succinic acid, tartaric acid, thiocyanic acid,p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and thelike.

“Base addition salt” refers to those salts which are prepared fromaddition of an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Salts derived from organic basesinclude, but are not limited to, salts of primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asammonia, isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Often crystallizations produce a solvate of the compound of theinvention. The present invention includes all solvates of the describedcompounds. As used herein, the term “solvate” refers to an aggregatethat comprises one or more molecules of a compound of the invention withone or more molecules of solvent. The solvent may be water, in whichcase the solvate may be a hydrate. Alternatively, the solvent may be anorganic solvent. Thus, the compounds of the present invention may existas a hydrate, including a monohydrate, dihydrate, hemihydrate,sesquihydrate, trihydrate, tetrahydrate and the like, as well as thecorresponding solvated forms. The compounds of the invention may be truesolvates, while in other cases the compounds of the invention may merelyretain adventitious water or another solvent or be a mixture of waterplus some adventitious solvent.

The compounds of the invention, or their salts, tautomers or solvatesmay contain one or more asymmetric centers and may thus give rise toenantiomers, diastereomers, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. The present invention is meant to includeall such possible isomers, as well as their racemic and optically pureforms. Optically active (+) and (−), (R)- and (S)-, or (D)- and(L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques, for example, chromatography andfractional crystallization. Conventional techniques for thepreparation/isolation of individual enantiomers include chiral synthesisfrom a suitable optically pure precursor or resolution of the racemate(or the racemate of a salt or derivative) using, for example, chiralhigh pressure liquid chromatography (HPLC). When the compounds describedherein contain olefinic double bonds or other centers of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present invention includestautomers of any said compounds. Various tautomeric forms of thecompounds are easily derivable by those of ordinary skill in the art.

The chemical naming protocol and structure diagrams used herein are amodified form of the I.U.P.A.C. nomenclature system, using the ACD/NameVersion 9.07 software program and/or ChemDraw Ultra Version 11.0software naming program (CambridgeSoft). Common names familiar to one ofordinary skill in the art are also used.

As noted above, in one embodiment of the present invention, compoundsuseful as fluorescent and/or colored dyes in various analytical methodsare provided. In general terms, embodiments of the present invention aredirected to dimers and higher polymers of fluorescent and/or coloredmoieties. The fluorescent and or colored moieties are linked by linkershaving multiple positively charged moieties or multiple negativelycharges moieties at the pH at which an assay is conducted. Withoutwishing to be bound by theory, it is believed electrostatic repulsion ofthe multiple charged moieties within the linker helps to maintainsufficient spatial distance between the fluorescent and/or coloredmoieties such that intramolecular quenching is reduced or eliminated,this resulting in a dye compound having a high molar “brightness” (e.g.,high fluorescence emission).

For example, the linker may comprise phosphate and/or thiophosphatemoieties when a negative charge is desired. When positive charges aredesired, linking groups containing quaternary amine groups and/or othergroups capable of holding a positive charge may be used. Accordingly, insome embodiments the compounds have the following structure (A):

wherein L is a linker comprising multiple positively charged moieties ormultiple negatively charged moieties, and the other variables are asdefined for structure (I). By “charged moieties” it is understood thatthe moieties will be charged at certain pH's, for example at the pH atwhich an assay employing the compound is performed, but it is not arequirement that the “charged moieties” be charged at all pH's.

In some other embodiments, the compounds have the following structure(I):

or a stereoisomer, tautomer or salt thereof, wherein:

M is, at each occurrence, independently a moiety comprising two or morecarbon-carbon double bonds and at least one degree of conjugation;

L¹, L² and L³ are, at each occurrence, independently an optionalalkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker;

L⁴ is, at each occurrence, independently an alkylene, alkenylene,alkynylene, heteroalkylene, heteroalkenylene or heteroalkynylene linker;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² and R³ are each independently H, OH, SH, alkyl, alkoxy, alkylether,—OP(═R_(a))(R_(b))R_(c), Q, a linker comprising a covalent bond to Q, alinker comprising a covalent bond to an analyte molecule, a linkercomprising a covalent bond to a solid support or a linker comprising acovalent bond to a further compound of structure (I), wherein: R_(a) isO or S; R_(b) is OH, SH, O⁻, S⁻, OR_(d) or SR_(d); R_(c) is OH, SH, O⁻,S⁻, OR_(d), SR_(d), alkyl, alkoxy, alkylether, alkoxyalkylether,phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether;

R⁴ is, at each occurrence, independently OH, SH, O⁻, S⁻, OR_(d) orSR_(d);

R⁵ is, at each occurrence, independently oxo, thioxo or absent;

Q is, at each occurrence, independently a moiety comprising a reactivegroup capable of forming a covalent bond with an analyte molecule, asolid support or a complementary reactive group Q′;

R^(d) is a cation;

m is, at each occurrence, independently an integer of zero or greater,provided that at least one occurrence of m is an integer of three orgreater; and

n is an integer of one or greater.

In some embodiments, m is, at each occurrence, independently an integerof three or greater.

In some embodiments, L¹, L² and L³ are, at each occurrence,independently optional alkylene or heteroalkylene linkers.

In some other embodiments of the compound of structure (I):

M is, at each occurrence, independently a moiety comprising two or morecarbon-carbon double bonds and at least one degree of conjugation;

L¹, L² and L³ are, at each occurrence, independently optional alkyleneor heteroalkylene linkers;

L⁴ is, at each occurrence, independently an alkylene, alkenylene,alkynylene, heteroalkylene, heteroalkenylene or heteroalkynylene linkerup to twenty atoms in length;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² and R³ are each independently H, OH, —OP(═R_(a))(R_(b))R_(c), Q, alinker comprising a covalent bond to Q, a linker comprising a covalentbond to an analyte molecule or a linker comprising a covalent bond to asolid support, wherein: R_(a) is O or S; R_(b) is OH, SH, O⁻, S⁻, OR_(d)or SR_(d); R_(c) is OH, SH, O⁻, S⁻, OR_(d), SR_(d), phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether;

R⁴ is, at each occurrence, independently OH, SH, O⁻, S⁻, OR_(d) orSR_(d);

R⁵ is, at each occurrence, independently oxo, thioxo or absent;

Q is a moiety capable of bonding with an analyte molecule or a solidsupport;

R_(d) is a cation;

m is, at each occurrence, independently an integer of zero or greater,provided that at least one occurrence of m is an integer of three orgreater; and

n is an integer of one or greater.

In some embodiments, the compound has the following structure (IA):

wherein x¹, x² and x³ are, at each occurrence, independently an integerfrom 0 to 6.

The L⁴ linker can be selected, along with other variables, to providethe desired fluorescence and/or color (“tune” the fluorescence and/orcolor). In some embodiments, L⁴ is a linker up to 20 atoms in length, upto 13 atoms in length, for example up to 10 atoms in length or up to 6atoms in length. In certain embodiments, L⁴ does not include disulfidebonds. In other embodiments, L⁴ is, at each occurrence, independentlyC₁-C₆ alkylene or C₂-C₆ alkynylene. In some embodiments, L⁴ is atwo-carbon linker.

In some other different embodiments, the compound has the followingstructure (IB):

wherein:

x¹, x² and x³ are, at each occurrence, independently an integer from 0to 6; and

y is, at each occurrence, independently an integer from 1 to 6.

In certain embodiments of the foregoing y is 2. In other embodiments,x¹, x² and x³ are each 1 at each occurrence. In some differentembodiments, x² is 0 and x³ is 1 at each occurrence.

In still other embodiments, R⁴ is, at each occurrence, independently OH,O⁻ or OR_(d). It is understood that “OR_(d)” and “SR_(d)” are intendedto refer to O⁻ and S⁻ associated with a cation. For example, thedisodium salt of a phosphate group may be represented as:

where R^(d) is sodium (Na⁺).

In further of the foregoing embodiments, R⁵ is, at each occurrence, oxo.

In some other embodiments, the compound has one of the followingstructures (IB′) or (IB″):

In any embodiments of the foregoing, R¹ is H.

In different embodiments, R² and R³ are each independently OH or—OP(═R_(a))(R_(b))R_(c). For example, in some embodiments R² and R³ areeach independently OH or —OP(═R_(a))(R_(b))R_(c), wherein R_(a) is O,R_(b) is OH, O⁻ or OR^(d); R_(c) is OH, O⁻, OR_(d), phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether; and R^(d) is a counter ion.

In still other different embodiments, one of R² or R³ is OH or—OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ is Q or a linkercomprising a covalent bond to Q. For example, in some embodiments one ofR² or R³ is OH or —OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ isQ or a linker comprising a covalent bond to Q, wherein R^(a) is O, R_(b)is OH, O⁻ or OR^(d); R_(c) is OH, O⁻, OR^(d), phosphate, thiophosphate,phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether; and R^(d) is a counter ion.

In still other embodiments, Q is or comprises a moiety capable ofbonding with an analyte molecule or a solid support. In certainembodiments, Q provides a means of connecting the compound of structure(I) to an analyte molecule or a solid support (e.g., by a covalentbond). For example, in some embodiments Q is or comprises a reactivegroup capable of forming a covalent bond with an analyte molecule or asolid support. In this regard the type of Q group and connectivity ofthe Q group to the remainder of the compound of structure (I) is notlimited. In certain embodiments, the Q is a moiety which is notsusceptible to hydrolysis under aqueous conditions, but is sufficientlyreactive to form a bond with a corresponding group on an analytemolecule or solid support (e.g., an amine).

Certain embodiments of compounds of structure (I) comprises Q groupscommonly employed in the field of bioconjugation. For example in someembodiments, Q is or comprises a nucleophilic reactive group, anelectrophilic reactive group or a cycloaddition reactive group. In somemore specific embodiments, Q is or comprises sulfhydryl, disulfide,activated ester, isothiocyanate, azide, alkyne, alkene, diene,dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide,biotin, amino or a maleimide. In some embodiments, the activated esteris an N-succinimide ester, imidoester or polyflourophenyl ester. Inother embodiments, the alkyne is an alkyl azide or acyl azide.

Exemplary Q moieties are provided in Table I below.

TABLE 1 Exemplary Q Moieties Structure Class

Sulfhydryl

Isothiocyanate

Imidoester

Acyl Azide

Activated Ester

Activated Ester

Activated Ester

Activated Ester

Activated Ester

Sulfonyl halide

Maleimide

Maleimide

α-haloimide

Disulfide

Phosphine

Azide

Alkyne

Biotin

Diene

Alkene/ dienophile

Alkene/ dienophile —NH₂ Amino

It should be noted that in some embodiments, wherein Q is SH, the SHmoiety will tend to form disulfide bonds with another sulfhydryl groupon another compounds of structure (I). Accordingly, some embodimentsinclude compounds of structure (I), which are in the form of disulfidedimers, the disulfide bond being derived from SH Q groups.

In some other embodiments, one of R² or R³ is OH or—OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ is a linkercomprising a covalent bond to an analyte molecule or a linker comprisinga covalent bond to a solid support. In some different embodiments, oneof R² or R³ is OH or —OP(═R_(a))(R_(b))R_(c), and the other of R² or R³is a linker comprising a covalent bond to an analyte molecule or alinker comprising a covalent bond to a solid support, wherein R^(a) isO, R_(b) is OH, O⁻ or OR^(d); R is OH, O⁻, OR^(d), phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether; and R^(d) is a counter ion. For example, in someembodiments the analyte molecule is a nucleic acid, amino acid or apolymer thereof. In other embodiments, the analyte molecule is anenzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer orprion. In still different embodiments, the solid support is a polymericbead or nonpolymeric bead.

The value form is another variable that can be selected based on thedesired fluorescence and/or color intensity. In some embodiments, m is,at each occurrence, independently an integer from 3 to 10. In otherembodiments, m is, at each occurrence, independently an integer from 7to 9.

The fluorescence intensity can also be tuned by selection of differentvalues of n. In certain embodiments, n is an integer from 1 to 100. Inother embodiments, n is an integer from 1 to 10.

M is selected based on the desired optical properties, for example basedon a desired color and/or fluorescence emission wavelength. In someembodiments, M is the same at each occurrence; however, it is importantto note that each occurrence of M need not be an identical M, andcertain embodiments include compounds wherein M is not the same at eachoccurrence. M may be attached to the remainder of the molecule from anyposition (i.e., atom) on M. One of skill in the art will recognize meansfor attaching M to the remainder of molecule.

In some embodiments, M is a fluorescent or colored moiety. Anyfluorescent and/or colored moiety known in the art and typicallyemployed in colorimetric, UV, and/or fluorescent assays may be used.Examples of M moieties which are useful in various embodiments of theinvention include, but are not limited to: Xanthene derivatives (e.g.,fluorescein, rhodamine, Oregon green, eosin or Texas red); Cyaninederivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine,thiacarbocyanine or merocyanine); Squaraine derivatives andring-substituted squaraines, including Seta, SeTau, and Square dyes;Naphthalene derivatives (e.g., dansyl and prodan derivatives); Coumarinderivatives; oxadiazole derivatives (e.g., pyridyloxazole,nitrobenzoxadiazole or benzoxadiazole); Anthracene derivatives (e.g.,anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange); Pyrenederivatives such as cascade blue; Oxazine derivatives (e.g., Nile red,Nile blue, cresyl violet, oxazine 170); Acridine derivatives (e.g.,proflavin, acridine orange, acridine yellow); Arylmethine derivatives:auramine, crystal violet, malachite green; and Tetrapyrrole derivatives(e.g., porphin, phthalocyanine or bilirubin). Other exemplary M moietiesinclude: Cyanine dyes, xanthate dyes (e.g., Hex, Vic, Nedd, Joe or Tet);Yakima yellow; Redmond red; tamra; texas red and Alexa Fluor® dyes.

In still other embodiments of any of the foregoing, M comprises three ormore aryl or heteroaryl rings, or combinations thereof, for example fouror more aryl or heteroaryl rings, or combinations thereof, or even fiveor more aryl or heteroaryl rings, or combinations thereof. In someembodiments, M comprises six aryl or heteroaryl rings, or combinationsthereof. In further embodiments, the rings are fused. For example insome embodiments, M comprises three or more fused rings, four or morefused rings, five or more fused rings, or even six or more fused rings.

In some embodiments, M is cyclic. For example, in some embodiments M iscarbocyclic. In other embodiment, M is heterocyclic. In still otherembodiments of the foregoing, M, at each occurrence, independentlycomprises an aryl moiety. In some of these embodiments, the aryl moietyis multicyclic. In other more specific examples, the aryl moiety is afused-multicyclic aryl moiety, for example which may comprise at least3, at least 4, or even more than 4 aryl rings.

In other embodiments of any of the foregoing compounds of structure (I),M, at each occurrence, independently comprises at least one heteroatom.For example, in some embodiments, the heteroatom is nitrogen, oxygen orsulfur.

In still more embodiments of any of the foregoing, M, at eachoccurrence, independently comprises at least one substituent. Forexample, in some embodiments the substituent is a fluoro, chloro, bromo,iodo, amino, alkylamino, arylamino, hydroxy, sulfhydryl, alkoxy,aryloxy, phenyl, aryl, methyl, ethyl, propyl, butyl, isopropyl, t-butyl,carboxy, sulfonate, amide, or formyl group.

In some even more specific embodiments of the foregoing, M, at eachoccurrence, independently is a dimethylaminostilbene, quinacridone,fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine,terrylene, sexiphenyl, porphyrin, benzopyrene,(fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl,(bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl,bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl,squaraine, squarylium, 9,10-ethynylanthracene or ter-naphthyl moiety. Inother embodiments, M¹ is, at each occurrence, independently p-terphenyl,perylene, azobenzene, phenazine, phenanthroline, acridine,thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, orperylene amide or a derivative thereof. In still more embodiments, M is,at each occurrence, independently a coumarin dye, resorufin dye,dipyrrometheneboron difluoride dye, ruthenium bipyridyl dye, energytransfer dye, thiazole orange dye, polymethine orN-aryl-1,8-naphthalimide dye.

In still more embodiments of any of the foregoing, M at each occurrenceis the same. In other embodiments, each M is different. In still moreembodiments, one or more M is the same and one or more M is different.

In some embodiments, M is pyrene, perylene, perylene monoimide or 6-FAMor derivative thereof. In some other embodiments, M¹ has one of thefollowing structures:

In some other embodiments, M¹ has the following structure:

In some specific embodiments, the compound is a compound selected fromTable 2:

TABLE 2 Exemplary Compounds Name Structure FC₃F

FC₄F

FC₅F

FC₆F

FC₇F

FC₈F

FC₉F

YC₃Y

YC₄Y

YC₅Y

YC₆Y

YC₃FC₃Y

YC₄FC₄Y

F(C₄F)₂

F(C₄F)₃

F(C₄F)₄

F(C₄F)₅

F(C₇F)₂

F(C₇F)₃

F(C₇F)₄

F(C₇F)₅

F(C₇F)₉

F(C₁₀F)₉

F(C₁₀F)₉SH

EC₃E

EC₄E

EC₅E

EC₆E

EC₇E

EC₈E

EC₉E

EC₁₀E

E(C₆E)₂

E(C₇E)₂

E(C₈E)₂

E(C₉E)₂

EC₃F

EC₄F

EC₅F

EC₆F

As used in Table 2, and throughout the application, F, E and Y refer tofluorescein, perylene and pyrene moieties, respectively, and have thefollowing structures:

The presently disclosed dye compounds are “tunable,” meaning that byproper selection of the variables in any of the foregoing compounds, oneof skill in the art can arrive at a compound having a desired and/orpredetermined molar fluorescence (molar brightness). The tunability ofthe compounds allows the user to easily arrive at compounds having thedesired fluorescence and/or color for use in a particular assay or foridentifying a specific analyte of interest. Although all variables mayhave an effect on the molar fluorescence of the compounds, properselection of M, m, n and L⁴ is believed to play an important role in themolar fluorescence of the compounds. Accordingly, in one embodiment isprovided a method for obtaining a compound having a desired molarfluorescence, the method comprising selecting an M moiety having a knownfluorescence, preparing a compound of structure (I) comprising the M,and selecting the appropriate variables for m, n and L⁴ to arrive at thedesired molar fluorescence.

Molar fluorescence in certain embodiments can be expressed in terms ofthe fold increase or decrease relative to the fluorescence emission ofthe parent fluorophore (e.g., monomer). In some embodiments the molarfluorescence of the present compounds is 1.1×, 1.5×, 2×, 3×, 4×, 5×, 6×,7×, 8×, 9×10× or even higher relative to the parent fluorophore. Variousembodiments include preparing compounds having the desired fold increasein fluorescence relative to the parent fluorophore by proper selectionof m, n and L⁴.

For ease of illustration, various compounds comprising phosphorousmoieties (e.g., phosphate and the like) are depicted in the anionicstate (e.g., —OPO(OH)O⁻, —OPO₃ ²⁻). One of skill in the art will readilyunderstand that the charge is dependent on pH and the uncharged (e.g.,protonated or salt, such as sodium or other cation) forms are alsoincluded in the scope of the invention.

Compositions comprising any of the foregoing compounds and one or moreanalyte molecules (e.g., biomolecules) are provided in various otherembodiments. In some embodiments, use of such compositions in analyticalmethods for detection of the one or more analyte molecules are alsoprovided.

In still other embodiments, the compounds are useful in variousanalytical methods. For example, in certain embodiments the disclosureprovides a method of staining a sample, the method comprising adding tosaid sample a compound of structure (I), wherein one of R² or R³ is alinker comprising a covalent bond to an analyte molecule (e.g.,biomolecule) or microparticle, and the other of R² or R³ is H, OH,phosphate or thiophosphate, in an amount sufficient to produce anoptical response when said sample is illuminated at an appropriatewavelength.

In some embodiments of the foregoing methods, R² is a linker comprisinga covalent linkage to an analyte molecule, such as a biomolecule. Forexample, a nucleic acid, amino acid or a polymer thereof (e.g.,polynucleotide or polypeptide). In still more embodiments, thebiomolecule is an enzyme, receptor, receptor ligand, antibody,glycoprotein, aptamer or prion.

In yet other embodiments of the foregoing method, R² is a linkercomprising a covalent linkage to a solid support such as amicroparticle. For example, in some embodiments the microparticle is apolymeric bead or nonpolymeric bead.

In even more embodiments, said optical response is a fluorescentresponse.

In other embodiments, said sample comprises cells, and some embodimentsfurther comprise observing said cells by flow cytometry.

In still more embodiments, the method further comprises distinguishingthe fluorescence response from that of a second fluorophore havingdetectably different optical properties.

In other embodiments, the disclosure provides a method for visuallydetecting an analyte molecule, such as a biomolecule, comprising:

-   -   (a) providing a compound of structure (I), wherein one of R² or        R³ is a linker comprising a covalent bond to the analyte        molecule, and the other of R² or R³ is H, OH, phosphate or        thiophosphate; and    -   (b) detecting the compound by its visible properties.

In some embodiments the analyte molecule is a nucleic acid, amino acidor a polymer thereof (e.g., polynucleotide or polypeptide). In stillmore embodiments, the analyte molecule is an enzyme, receptor, receptorligand, antibody, glycoprotein, aptamer or prion.

In other embodiments, a method for visually detecting an analytemolecule, such as a biomolecule is provided, the method comprising:

-   -   (a) admixing any of the foregoing compounds with one or more        analyte molecules; and    -   (b) detecting the compound by its visible properties.

In some other different embodiments, the compounds of structure (I) canbe used in various methods for analysis of cells. For example, by use offlow cytometry, the compounds can be used to discriminate between liveand dead cells, evaluate the health of cells (e.g., necrosis vs. earlyapoptitic vs. late apoptitic vs. live cell), tracking ploidy and mitosisduring the cell cycle and determining various states of cellproliferation. While not wishing to be bound by theory, it is believedthat embodiments of the compounds of structure (I) preferentially bindor associate with positively charged moieties. Accordingly, in someembodiments the compounds may be used in methods for determining thepresence of non-intact cells, for example necrotic cells. For example,in some embodiments is provided a method for determining the presence ofnecrotic cells, the method comprising admixing a sample containing cellswith a compound of structure (I) and analyzing the mixture by flowcytometry. The compound of structure (I) binds or associates withnecrotic cells, and thus the presence of necrotic cells is detectableunder flow cytometry conditions. In contrast to other staining reagentswhich require an amine reactive group (or other reactive group) to bindto necrotic cells, embodiments of the staining methods employingcompounds of structure (I) do not require a protein-free incubationbuffer, and thus the methods are more efficient to perform than relatedknown methods.

Accordingly, in some embodiments the invention provides a method fordetermining the presence of dead cells (or non-intact cells or necrotictissue, etc.) in a sample, the method comprising contacting the samplewith a compound of structure (I), thereby binding or associating thecompound with the dead cells, and observing a fluorescent signal fromthe compound bound or associated with the dead cells. For example, someembodiments comprise use of flow cytometry to observe the compound boundor associated with the dead cells. As noted above, certain methods donot require use of reactive groups to bind or associate with the deadcells. Accordingly, in certain embodiments the compound of structure (I)for use in identification of dead cells is a compound of structure (I),wherein R² and R³ are each independently OH or —OP(═R_(a))(R_(b))R_(c).

In various other embodiments, the compounds can be used in relatedmethods for determine the presence of positively charged moieties inintact or non-intact cells, apoptitic bodies, depolarized membranesand/or permealized membranes.

In addition to the above methods, embodiments of the compounds ofstructure (I) find utility in various disciplines and methods, includingbut not limited to: imaging in endoscopy procedures for identificationof cancerous and other tissues; identification of necrotic tissue bypreferential binding of the compounds to dead cells; single-cell and/orsingle molecule analytical methods, for example detection ofpolynucleotides with little or no amplification; cancer imaging, forexample by conjugating a compound of structure (I) to an antibody orsugar or other moiety that preferentially binds cancer cells; imaging insurgical procedures; binding of histones for identification of variousdiseases; drug delivery, for example by replacing the M moiety in acompound of structure (I) with an active drug moiety; and/or contrastagents in dental work and other procedures, for example by preferentialbinding of the compound of structure (I) to various flora and/ororganisms.

It is understood that any embodiment of the compounds of structure (I),as set forth above, and any specific choice set forth herein for a R¹,R², R³, R⁴, R⁵, L¹, L², L³, L⁴, M, m and n variable in the compounds ofstructure (I), as set forth above, may be independently combined withother embodiments and/or variables of the compounds of structure (I) toform embodiments of the inventions not specifically set forth above. Inaddition, in the event that a list of choices is listed for anyparticular R¹, R², R³, R⁴, R⁵, L¹, L², L³, L⁴, M, m and n variable in aparticular embodiment and/or claim, it is understood that eachindividual choice may be deleted from the particular embodiment and/orclaim and that the remaining list of choices will be considered to bewithin the scope of the invention.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

It will also be appreciated by those skilled in the art that in theprocess described herein the functional groups of intermediate compoundsmay need to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

Furthermore, all compounds of the invention which exist in free base oracid form can be converted to their salts by treatment with theappropriate inorganic or organic base or acid by methods known to oneskilled in the art. Salts of the compounds of the invention can beconverted to their free base or acid form by standard techniques.

The following Reaction Schemes illustrate exemplary methods of makingcompounds of this invention. It is understood that one skilled in theart may be able to make these compounds by similar methods or bycombining other methods known to one skilled in the art. It is alsounderstood that one skilled in the art would be able to make, in asimilar manner as described below, other compounds of structure (I) notspecifically illustrated below by using the appropriate startingcomponents and modifying the parameters of the synthesis as needed. Ingeneral, starting components may be obtained from sources such as SigmaAldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI,and Fluorochem USA, etc. or synthesized according to sources known tothose skilled in the art (see, for example, Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, 5th edition (Wiley, December2000)) or prepared as described in this invention.

Reaction Scheme I illustrates an exemplary method for preparing anintermediate useful for preparation of compounds of structure (I), whereR¹, L¹, L², L³ and M are as defined above, and R² and R³ are as definedabove or are protected variants thereof. Referring to Reaction Scheme 1,compounds of structure a can be purchased or prepared by methodswell-known to those of ordinary skill in the art. Reaction of a withM-X, where x is a halogen such as bromo, under Suzuki couplingconditions known in the art results in compounds of structure b.Compounds of structure b can be used for preparation of compounds ofstructure (I) as described below.

Reaction Scheme II illustrates an alternative method for preparation ofcompounds of intermediates useful for preparation of compounds ofstructure (I). Referring to reaction Scheme II, where R¹, L¹, L², L³ andM are as defined above, and R² and R³ are as defined above or areprotected variants thereof, a compound of structure c, which can bepurchased or prepared by well-known techniques, is reacted with M-Z toyield compounds of structure d. Here, Y and Z represent functionalgroups having complementary reactivity (i.e., functional groups whichreact to form a covalent bond). Z may be pendant to M or a part of thestructural backbone of M, for example a cyclic anhydride. Y may be anynumber of functional groups, such as amino.

In certain embodiments, the compounds of structure I are oligomerscomprising from 2-100 repeating units. Such oligomers can be preparedusing methods analogous to well-known automated DNA synthesis methods.DNA synthesis methods are well-known in the art. Briefly, two alcoholgroups, for example R² and R³ in intermediates b or d above, arefunctionalized with a dimethoxytrityl (DMT) group and a2-cyanoethyl-N,N-diisopropylamino phosphoramidite group, respectively.The phosphoramidite group is coupled to an alcohol group, typically inthe presence of an activator such as tetrazole, followed by oxidation ofthe phosphorous atom with iodine. The dimethoxytrityl group can beremoved with acid (e.g., chloroacetic acid) to expose the free alcohol,which can be reacted with a phosphoramidite group. The 2-cyanoethylgroup can be removed after oligomerization by treatment with aqueousammonia.

Preparation of the phosphoramidites used in the oligomerization methodsis also well-known in the art. For example, a primary alcohol (e.g., R³)can be protected as a DMT group by reaction with DMT-Cl. A secondaryalcohol (e.g., R²) is then functionalized as a phosphoramidite byreaction with an appropriate reagent such as 2-cyanoethylN,N-dissopropylchlorophosphoramidite. Methods for preparation ofphosphoramidites and their oligomerization are well-known in the art anddescribed in more detail in the examples.

Oligomers of intermediates b or d are prepared according to thewell-known phophoramidite chemistry described above. The desired numberof m repeating units is incorporated into the molecule by repeating thephosphoramidite coupling the desired number of times with an appropriateintermediate, for example an intermediate having the followingstructure:

The following examples are provided for purposes of illustration, notlimitation.

EXAMPLES

General Methods

¹H and ³¹P NMR spectra were obtained on a JEOL 400 MHz spectrometer. ³¹PNMR spectra were referenced against 85% aqueous phosphoric acid and ¹Hspectra were referenced against TMS. Reverse phase HPLC dye analysis wasperformed using a Waters Acquity UHPLC system with a 2.1 mm×50 mmAcquity BEH-C18 column held at 45° C. Mass spectral analysis wasperformed on a Waters/Micromass Quattro micro MS/MS system (in MS onlymode) using MassLynx 4.1 acquisition software. Mobile phase used forLC/MS on dyes was 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 8.6mM triethylamine (TEA), pH 8. Phosphoramidites and precursor moleculeswere analyzed using an Agilent Infinity 1260 UHPLC system with a diodearray detector and High Performance Autosampler using an Aapptec®Spirit™ Peptide C18 column (4.6 mm×100 mm, 5 m particle size).Excitation and emission profiles experiments were recorded on a CaryEclipse spectra photometer.

All reactions were carried out in oven dried glassware under a nitrogenatmosphere unless otherwise stated. Commercially available DNA synthesisreagents were purchased from Glen Research (Sterling, Va.). Anhydrouspyridine, toluene, dichloromethane, diisopropylethyl amine,triethylamine, acetic acid, pyridine, and THF were purchased fromAldrich. All other chemicals were purchased from Aldrich or TCI and wereused as is with no additional purification.

All oligomer (i.e., dimer and higher) dyes were synthesized on an ABI394 DNA synthesizer using standard protocols for thephosphoramidite-based coupling approach. The chain assembly cycle forthe synthesis of oligomers was the following: (i) detritylation, 3%trichloroaceticacid in dichloromethane, 1 min; (ii) coupling, 0.1 Mphosphoramidite and 0.45 M tetrazole in acetonitrile, 10 min; (iii)capping, 0.5 M acetic anhydride in THF/lutidine, 1/1, v/v 15 s; (iv)oxidation, 0.1 M iodine in THF/pyridine/water, 10/10/1, v/v/v, 30 s.

Chemical steps within the cycle were followed by acetonitrile washingand flushing with dry argon for 0.2-0.4 min. Cleavage from the supportand removal of base and phosphoramidate protecting groups was achievedby treatment with ammonia for 1 hour at room temperature. Oligomer dyeswere then analyzed by reverse phase HPLC as described above.

Example 1 Synthesis of Phosphoramidite Dye Monomers

1-O-(4,4′-dimethoxytrityl-2-methylene-1,3-propanediol(1)

Into a dry 500 mL round bottom flask was put a stir bar. After flushingwith nitrogen, dry pyridine (240 mL) was added, and the flask was cooledin an ice bath for 15 minutes. Upon cooling DMTrCl (7.65 g, 22.5 mmol)was added after which the flask was stirred overnight in a refrigeratorat 4° C. under a nitrogen atmosphere. Several drops of methanol werethen added and the reaction was concentrated in vacuo to a viscous gum.The resulting gum was dissolved in EtOAc (200 mL) and washed with NaHCO₃(250 mL) and sat. NaCl (250 mL). The organic layer was dried over Na₂SO₄and concentrated in vacuo to a viscous gum. The isolated crude productwash then purified by silica gel column chromatography eluting with agradient of EtOAc:hexanes (25:75 v/v)-(1:1 v/v) to give 1 as a clear gum(5.21 g, 60%). ¹H NMR was recorded and found to be consistent with thestructure of compound 1.

1-O-(4,4′-dimethoxytrityl)-2-hydroxymethyl-3-pyrenylpropanol(2)

Into a dry 250 mL round bottom flask fitted with a condenser was put astir bar. The flask was purged with nitrogen, and dry THF (40 mL) andcompound 1 (5.0 g, 12.8 mmol) were added. 0.5 M 9-BBN in THF (65 mL, 32mmol) was added via syringe and the reaction was heated to reflux for 12hrs. After allowing the reaction to cool to room temperature, 3M K₂CO₃(11 ml) and dry DMF (100 mL) were added. 1-Bromopyrene (2.0 g, 6.5 mmol)and PdCl₂(dppf) (0.65 g, 0.8 mmol) were added, and the solution wasallowed to stir for 15 hrs at room temperature. The reaction mixture waspoured into CH₂Cl₂ (300 mL) and washed with H₂O (500 mL). The aqueouslayer was then back extracted with additional CH₂Cl₂ (200 mL). Thecombined organic layers were washed with sat. NaCl (300 mL), dried overNa₂SO₄, and concentrated in vacuo to a viscous gum. The isolated crudeproduct wash then purified by silica gel column chromatography elutingwith a gradient of EtOAc:hexanes (25:75 v/v)-(1:1 v/v) to give 2 as aclear gum (3.0 g, 79%). The ¹H NMR spectrum was recorded and found to beconsistent with the structure of compound 2.

1-O-(4,4′-dimethoxytrityl)-2-methylpyrene-3-O-(2-cyanoethyl-N,N-diisopropyl)propane phosphoramidite (3)

Into a dry 100 mL round bottom flask was put a stir bar. After purgingthe flask with nitrogen, CH₂Cl₂ (20 mL) and compound 2 (0.30 g, 0.50mmol) were added. N,N-Diisopropylethylamine (0.88 mL, 5.0 mmol) and2-cyanoethyl diisopropychlorophosphoramidite (0.45 mL, 2.0 mmol) wereadded via syringe. After 1 hour of stirring at room temperature, thereaction was determined to be complete by TLC analysis. The crudereaction mixture was then purified directly by silica gel columnchromatography eluting with a gradient of EtOAc:hexanes:TEA (22.5:72.5:5v/v/v) to give 3 as a white foam (0.28 g, 70%). The ³¹P NMR spectrum wasrecorded and found to be consisted with the structure of compound 3:Purity was determined by HPLC analysis with detection at 254 and 340 nm.

Other compounds with different Ar groups (e.g., any of the “M” groupsdescribed herein) were prepared in an analogous manner.

Example 2 Synthesis of Perylene Carbodiimide Dye Monomer

N-(2,3-propanediol) perylenemonoimide(4)

Into a dry 200 mL round bottom flask fitted with a condenser was put astir bar and perylene monoanhydride¹ (1.83 g, 5.67 mmol). After adding3-amino-1,2-propanediol (1.1 g, 2.1 mmol) and imidazole (14.3 g, 0.21mol), the vessel was heated to 140° C. in an oil bath for 15 hours. Thereaction was allowed to cool to room temperature and then 10% HCl wasadded (500 mL). The resulting deep red precipitate was collected byfiltration, washed well with water and dried at 180° C. for severalhours to yield 4 as a deep red solid (1.95 g, 86%).

N-(3-O-(4,4′-dimethoxytrityl-2-hydroxypropane) perylenemonoimide(5)

Into a dry 200 mL round bottom flask was put a stir bar. After purgingthe flask with nitrogen, dry pyridine (120 mL), compound 4 (0.44 g, 1.1mmol), and dimethoxytritylchloride (0.45 g, 1.3 mmol) were all added,and the reaction was allowed to stir at room temperature for 48 hours.Several drops of methanol were then added, and the reaction wasconcentrated in vacuo to a viscous gum. The resulting gum was dissolvedin CH₂Cl₂ (200 mL) and washed with sat. NaCl (200 mL). The aqueous layerwas washed with in CH₂Cl₂ (3×100 mL). The combined organic layers weredried over Na₂SO₄ and concentrated in vacuo to a viscous gum. Theisolated crude product wash then purified by silica gel columnchromatography eluting with a gradient of EtOAc: CH₂Cl₂ (0:100 v/v)-(2:3v/v) to give 5 as a red foam (0.25 g, 50%).

N-(3-O-(4,4′-dimethoxytrityl-2-O-(2-cyanoethyl-N,N-diisopropylamidephosphoramidite) perylene-monoimide (6)

Into a dry 50 ma round bottom flask was put a stir bar. After purgingthe flask with nitrogen, CH₂Cl₂ (5 mL) and compound 5 (0.25 g, 0.36mmol) were added. N,N-diisopropylethylamine (0.24 mL, 1.79 mmol) and2-cyanoethyl N,N-diisopropychlorophosphoramidite (0.16 mL, 0.72 mmol)were added via syringe. After 1 hour of stirring at room temperature,the reaction was determined to be complete by TLC analysis. The crudereaction mixture was then purified directly by silica gel columnchromatography eluting with CH₂Cl₂:TEA (95:5 v/v) to give 6 as a darkred foam (0.26 g, 80%). The purified compound was analyzed by RP-HPLCwith observation at 254 and 500 nm. Two diastereomers were found to bepresent.

Other dye monomers with different M groups were prepared in an analogousmanner.

Example 3 Synthesis of Oligomer Dyes

Oligomer dyes were synthesized on an Applied Biosystems 394 DNA/RNAsynthesizer or on GE AKTÄ 10 OligoPilot on either 1 μmol or 10 μmolscales and possessed a 3′-phosphate group. Dyes were synthesizeddirectly on CPG beads or on polystyrene solid support. The dyes weresynthesized in the 3′ to 5′ direction by standard solid phase DNAmethods. Coupling methods employed standard 3-cyanoethyl phosphoramiditechemistry conditions. Different number of “m” repeating units wereincorporated by repeating the synthesis cycle the desired number oftimes with an appropriate phosphoramidite. All phosphoramidite monomerswere dissolved in acetonitrile/dichloromethane (0.1 M solutions), andwere added in successive order using the following synthesis cycles: 1)removal of the 5′-dimethoxytrityl protecting group with dichloroaceticacid in toluene, 2) coupling of the next phosphoramidite with activatorreagent in acetonitrile, 3) oxidation with iodine/pyridine/water, and 4)capping with acetic anhydride/1-methylimidizole/acetonitrile. Thesynthesis cycle was repeated until the 5′ Oligofloroside was assembled.At the end of the chain assembly, the monomethoxytrityl (MMT) group ordimthoxytrityl (DMT) group was removed with dichloroacetic acid indichloromethane or dichloroacetic acid in toluene.

The dyes were cleaved from the solid support and deprotected as follows:

A 1 mL micropipettor was used to add 450 μL of concentrated NH₄OH to ˜25mg of reacted CPG solid support in a 1.5 mL Eppendorf tube. The slurrywas mixed briefly using a Vortex mixer and allowed to settle beforeplacing (open) on a 55° C. heating block until gas formation (andbubbling) started to diminish, at which point the tube was tightlyclosed. Heat treatment was for 2 hours (+/−15 minutes) and tubes werethen removed to cool to room temperature. The tube and its contents werespun in a centrifuge at its maximum speed (13400 rpm) for 1 minute, andthen the supernatant was removed with a glass pipette and placed into asecond, labeled, 1.5 mL Eppendorf tube, taking care not to include thesupport. The support was washed and spun-down 2× with ˜150 μL ofacetonitrile to help maximize dye removal, and the washings werecarefully removed from support and added to the labeled secondary tubes.Clarified supernatant was dried completely in a CentriVap concentratorat 40° C. to remove NH₄OH.

Example 4 Characterization of Oligomer Dyes

1 mL of deionized water was added to the dried dye sequence preparedaccording to Example 3 to re-constitute and establish a concentratedstock of ˜0.3 to 1.0 mM (determined later). 2 μL aliquots of each dyeconstruct were analyzed by HPLC-MS to determine identity and relativepurity using 45° C. heated ultra-high performance 2.1 mm×50 mm C18column (1.7 μm) with 150 mM HFIP/TEA (pH9) mobile phase, and methanol asorganic elution component. Gradient was from 1-100% over 10 minutes.Electrospray ionization was used (in negative mode) to determine themolecular weights of the dye sequences and help to characterizeimpurities.

A sample was taken from a concentrated stock using a micropipettor anddiluted appropriately in 0.1×PBS (10× to 100×) to be within linear rangeof the NanoDrop UV-vis spetrophotomer (Thermo Scientific). A blankmeasurement was performed on the NanoDrop using 0.1×PBS, and then theabsorbance of the diluted dye sequence at an appropriate wavelength wasrecorded. Extinction coefficients (E) were determined by the totalnumber of fluors (M moieties) in the dye construct, using 75,000 M⁻¹ cm¹for each fluorescein (F; read at 494 nm); 34,500 for each pyrene (Y;read at 343 nm); and 40,000 for each perylene (E; read at 440 nm)present in the sequence. Spacers are presumed to have no effect on ε. Anexemplary calculation of the molar concentration of a dye having thestructure [F(CCCCCCCF)₅] is as follows:Molar concentration of dye={A ₄₉₄/(L*ε _(Dye))}*DilutionFactor  Equation 1:

-   -   E_(Dye)=450,000 M⁻¹ cm⁻¹    -   L_(nanodrop)=0.1 cm    -   A₄₉₄=0.254 AU    -   Dilution Factor=100    -   Molar concentration of Dye=5.64×10⁻⁰⁴ M, or 0.564 mM.

With concentration determined, the dye stock was diluted in the NaPO⁴(0.1 μM at pH 7.5) and NaCO³ (0.1 M at pH 9.0) buffers to make solutionsof 2 μM (or 5 μM, whatever works with the linear range of theinstrument) at a final volume of ˜3.5 mL. These solutions were scannedby UV/Vis, and then used them to make a second dilution in theappropriate buffer for reading on the fluorimeter, in the range of 10-50nM. The necessary concentration will vary depending upon the identity ofthe M moiety. For the above dye, a 25 nM solution was used

Using a 1 cm quartz cuvette, the absorbance of the 2 μM sample wasdetermined, scanning from 300 nm to 700 nm. Scan speed was set tomedium.

Using a 1 cm quartz cuvette and a Cary Eclipse spectrometer, theemission of the 25 nM sample was read using an appropriate excitationwavelength (494 nm for above dye) and scanning from 499 nm to 700 nm.Scan speed was set to medium.

Example 5 UV and Fluorescence Properties of Oligomer Dyes

The dye compounds listed in Table 3 were prepared according to the abovegeneral procedures and their UV and fluorescence properties weredetermined at 2 nM and pH 9. As can be seen from the UV spectra in FIG.1, the extinction coefficients of the various dye compounds roughlycorrespond to the theoretical value (i.e., the extinction coefficientsare additive).

The fluorescence emission spectra from 499 nm to 700 nm of the dyes inTable 3 was also determined (FIG. 2). In contrast to the extinctioncoefficients, the emission of the dyes containing multiple fluorescentmoieties was significantly less than the parent compound (F). While notwishing to be bound by theory, it is believed that this phenomenon canbe explained by intramolecular quenching of fluorescence by the multiplefluorescent moieties.

TABLE 3 Comparative Dye Compounds Name Structure ϵ_(t)* F F—OH  75k FCF

150k F(CF)₂

225k F(CF)₃

300k F(CF)₄

375k F(CF)₅

450k F(CF)₆

525k ϵ_(t) = theoretical molar extinction coefficient

Example 6 UV and Fluorescence Properties of Oligomer Dyes

To explore the effects of various linker lengths between the fluorescentmoieties, the dye compounds listed in Table 4 were prepared according tothe above general procedures and their UV and fluorescent propertiesdetermined.

The extinction coefficients of each of the fluorescein dimer dyes inTable 4 roughly corresponds with the expected theoretical value based onthe sum of the two fluorescein moieties (FIG. 3). Surprisingly, thefluorescent emission spectra of the dimers indicate that the linkerlength has a significant effect on the fluorescent emission of the dye(FIG. 4). Specifically, it was found that inclusion of at least “C”spacers (see structure below) between the fluorescein moieties resultedin an overall increase in emission relative to a single fluorescein,whereas shorter linkers between the fluorescein moieties resulted indecreased fluorescence relative to a single fluorescein.

TABLE 4 Dye Compounds Name Structure ϵ_(t)* F F—OH  75k FF

150k FCF

150k FC₂F

150k FC₃F

150k FC₄F

150k FC₅F

150k FC₆F

150k FC₇F

150k FC₈F

150k FC₉F

150kε_(t) and F are as defined above, and the “C linker” has the followingstructure:

Example 7 UV and Fluorescence Properties of Oligomer Dyes

To explore the effects of the number of fluorescent moieties within adye molecule, the dye compounds listed in Table 5 were preparedaccording to the above general procedures and their UV and fluorescentproperties determined at 2 nM and pH 9.

Again the extinction coefficients of each of the fluorescein dimer dyesin Table 5 was additive and roughly corresponds with the expectedtheoretical value (FIG. 5). The fluorescent emission of each of thecompounds in Table 5 is brighter than the parent (i.e., monomer)fluorophore (FIG. 6).

TABLE 5 Dye Compounds Name Structure ϵ_(t)* F F—OH  75k FC₄F

150k F(C₄F)₂

225k F(C₄F)₃

300k F(C₄F)₄

375k F(C₄F)₅

450kε_(t) and F are as defined above.

Example 8 UV and Fluorescence Properties of Oligomer Dyes

The dye compounds listed in Table 6 were prepared according to the abovegeneral procedures and their fluorescent emission properties determinedat 25 nM and pH 9.

The fluorescent emission spectra indicate that each of these compoundsare brighter than the parent (i.e., monomer) fluorophore, with onecompound being approximately 6× brighter (FIG. 7).

TABLE 6 Dye Compounds Name Structure ϵ_(t)* F F—OH  75k FC₇F

150k F(C₇F)₂

225k F(C₇F)₃

300k F(C₇F)₄

375k F(C₇F)₅

450kε_(t) and F are as defined above.

Example 9 UV and Fluorescence Properties of Oligomer Dyes

The compounds in Table 7 were prepared according to the above generalprocedures and their fluorescent emission properties determined at 10 nMand pH 9.

The fluorescent emission spectra of these compounds again show thatthese compounds are brighter than the parent (i.e., F) fluorophore (FIG.8, unity value not shown).

TABLE 7 Dye Compounds Name Structure F F—OH FC₇F

F(C₇F)₂

F(C₇F)₃

F(C₇F)₄

F(C₇F)₅

F(C₇F)₉

F is as defined above.

Example 10 UV and Fluorescence Properties of Oligomer Dyes ContainingPerylene

Dye compounds comprising a perylene moiety (“E”) were prepared accordingto the above general procedures and their fluorescent emissionproperties determined at 50 nM and pH 9. The structure of these dyes ispresented in Table 8.

The fluorescent emission spectra indicate that each of these compoundsare brighter than the parent (i.e., “unity”) fluorophore (FIG. 9). Theability to use perylene in an aqueous environment, and increase itsbrightness on a molar basis, is a major advance given the hydrophobicnature of this fluorophore.

TABLE 8 Perylene Containing Dye Compounds Name Structure E

E(C₆E)₂

E(C₇E)₂

E is as defined above.

Example 11 General Flow Cytometry Method and Applications

The general flow cytometry workflow includes the following steps:

1. Culture and visually observe cells for signs of metabolic stressand/or use fresh, induced, or simulated cells.

2. Dilute dye compounds to working volumes.

3. Harvest and prepare cells without killing or inducing apoptosis.

4. Centrifuge and wash cells with appropriate buffer.

5. Perform cell counts using hemocytometer and trypan blue exclusion.

6. Centrifuge and wash cells

7. Adjust cell density to test size

8. Apply dye (pre-dilution) or other co-stains of interest.

9. Incubate the cell/stain/dye mixture.

10. Centrifuge and wash cells with appropriate buffer.

11. Re-suspend cells in acquisition buffer.

12. Acquire cell data by flow cytometry.

The general workflow described above can be modified according tospecific applications. Some modifications for specific applications aredescribed below.

Live/Dead Discrimination

Cells are tested for viability by positively staining necrotic cells tocompare damaged cells to intact cells. Assays are used to targetnon-intact (fixed and non-fixed) cells with positively charged moieties,cell debris, apoptotic bodies, depolarized cell membrane, andpermeabilized membranes. Cells are then stained with dye using routinecell preparations (fresh or fixed) and analyzed using flow cytometry.

Cell Health

A comparison is made between dead cells (i.e., necrotic cells), earlyapoptotic, late apoptotic, and live cells. Dead cells are positivelystained, Apoprotic bodies are intermediately stained, and live cells areleft negative. This strategy results in very bright necrotic cells andworks also to assess cell permeability. Assays are used to targetnon-intact (fixed and non-fixed) cells with positively charged moieties,cell debris, apoptotic bodies, depolarized cell membrane, andpermeabilized membranes. Dye staining is performed on in vitro cultures,primary cells, and samples treated with xenobiotics and analyzed usingflow cytometry.

Cell Cycle

Cell ploidy and mitosis in the cell cycle is tracked by stainingcorrelated to positively staining DNA intercalators in all cells andcellular bodies containing nucleic acid and cell cycle associatedproteins. Assays are used to target non-intact (non-fixed only) cellswith positively charged moieties, cell debris, apoptotic bodies,depolarized cell membrane, and permeabilized membranes. Assays are usedto target intact (fixed and permeabilized) cells by staining positivelycharged moieties after preservation of cells are fixed and permeabilizedfor intracellular staining. Dye staining (in combination with otherdyes) is performed on in vitro cultures, primary cells, and samplestreated with xenobiotics and analyzed using flow cytometry.

Proliferation

Cell proliferation is monitored by staining correlated to positivelystaining DNA intercalators in all cells and cellular bodies containingnucleic acid and cell cycle associated proteins. Assays are used totarget non-intact (non-fixed only) cells with positively chargedmoieties, cell debris, apoptotic bodies, depolarized cell membrane, andpermeabilized membranes. Assays are used to target intact (fixed andpermeabilized) cells by staining positively charged moieties afterpreservation of cells are fixed and permeabilized for intracellularstaining. Dye staining (in combination with monitoring markers for cellproliferation, e.g. Ki67, BRDU) is performed on in vitro cultures,primary cells, and samples treated with xenobiotics and analyzed usingflow cytometry.

Example 12 Cell Culture of Jurkat Cells

Jurkat cells (Clone E6-1; ATCC® TIB-152™) are human lymphocyte cellsfound in peripheral blood tissue and used to model acute T cellLeukemia. Cells were cultured in RPMI-1640 Medium, fetal bovine serum10%, 0.1 M HEPES, PenStrep and L-glutamine.

Cultures were maintained by addition of fresh medium or replacement ofmedium between 1×10⁵ viable cells/mL and 5×10⁶ cells/mL. Alternatively,cultures were established by centrifugation with subsequent resuspensionat 1×10⁵ viable cells/mL. Fresh medium was added every 2 to 3 daysdepending on cell density.

Example 13 Cell Culture of Ramos Cells

Ramos (RA 1; ATCC CRL-1596) are human B lymphocytes and are used tomodel Burkitt's Lymphoma (American). Cells were cultured in RPMI-1640Medium with heat-inactivated fetal bovine serum 10%, 0.1 HEPES, PenStrepand L-glutamine.

Cultures were maintained by addition of fresh medium or replacement ofmedium between 1×10⁵ viable cells/mL and 5×10⁶ cells/mL. Alternatively,cultures were established by centrifugation with subsequent resuspensionat 1×10⁵ viable cells/mL. Fresh medium was added every 2 to 3 daysdepending on cell density.

Example 14 General Procedure for Inducing Cell Death and Recovering DeadCells

Cultured cells were induced into necrosis and apoptosis in vitro to formdead cells, cell debris, and apoptotic bodies. Cells were induced byintroducing heat stress and/or metabolic stress. Heat stress wasperformed by subjecting cultures to 57-60° C. for 3-5 minutes and thentransferring cell cultures to ice for 10 minutes. Metabolic stress wasperformed by post log phase growth crowding, toxin, or xenobiotictreatment (e.g. 10 nM maptothecin). Fresh cells were maintained bysubjecting cultures to no treatment.

Cell preparations were re-suspended in staining buffer, and then mixedin ratios to target a viable population of intact cells between 60-80%(i.e., 20-40% non-intact cellular debris-like events as measured bymorphology using flow cytometry parameters FSC v. SSC). Cell viabilitywas verified microscopically by Live/Dead trypan blue exclusion or by7-aminoactinomycin D (7-AAD) by flow cytometry. In some experiments,mixtures of induced/non-induced cells were unnecessary. Instead, apost-log phase growth culture undergoing metabolic stress containing30-40% necrotic and apoptotic cells was used.

Example 15 Inducing Cell Death and Recovery of Dead Cells for Jurkat andRamos Cells

Mid-log phase Jurkat and Ramos or primary PBMC cells were induced intonecrosis in vitro in order to form dead cells and cell debris. Cellswere induced by heat stress by subjecting cultured cells first to 58° C.for 3-5 minutes, then transferring them to ice for 10-15 minutes. Freshcells were maintained by subjecting cultures to no treatment.

Cells were resuspended in 1×DBS with Ca⁺⁺/Mg⁺⁺ at a cell density of10×10⁶ cells/mL. Fresh cells (˜8% necrotic) and stressed cells (˜20%necrotic) were then mixed in the following ratios to produce ameasurable gradient of viable to necrotic cells:

1. 100% live:0% dead

2. 50% live:50% dead

3. 25% live:75% dead

4. 12.75% live:85.25% dead

5. 6% live:94% dead

The expected viability ranged from 8-30%. Starting and endingviabilities were verified by 7-Aminoactinomycin D (7-AAD) by flowcytometry or typan clue exclusion.

Example 16 Reaction of Dead, Necrotic, and Apoptotic Cells with F(C₁₀F)₉and F(C₁₀F)₉SH Dyes

Cells and dye (e.g., bulk F(C₁₀F)₉ or HNSA activated, amine reactiveF(C₁₀F)₉SH, see Table 2) were reacted together in 96 well U-bottompolypropylene plates. Dyes were diluted serially in pure deionized wateror sterile filtered phosphate buffered solution to a concentration rangeof 15,000-0.001 μM in volumes of 25-200 μL. Phosphate buffered salinewas used for amine reactive dye (i.e. F(C₁₀F)₉SH). The dye solutionswere then applied to cell preparations. Dyes diluted in water werelimited to test volumes of ≤25 μL as applied to cells. Cell density wasmaintained at 2-3×10⁶ cells/mL per test well (V_(f range)=100-200 μLstain buffer containing protein and metabolic inhibitors or phosphatebuffered saline).

Prior to incubation with the dyes, cells were bulk washed (15-50 mLconical tubes) two or more times in complete growth medium (first wash),and wash buffer containing protein, phosphate buffered saline, andmetabolic inhibitors in ratios of 2-3×10⁵ cells/test per mL of wash(later washes). Large wash volume centrifuged (g×500) for 10 minutes.When F(C₁₀F)₉SH was used, cells were resuspended in phosphate bufferedsaline before being stained with dye.

The dye mixtures were then incubated for 30-40 minutes at 23-25° C.After incubation, cells were washed twice with buffer (containingprotein, phosphate buffered saline, and metabolic inhibitors) by theaddition of 600-800 L/well. Mixtures were then centrifuged (g×350) for 5minutes.

The data in FIGS. 10a and 10b show that staining of necrotic cells isdose dependent with respect to the concentration of dye (e.g.,F(C₁₀F)₉SH or F(C₁₀F)₉) used.

Example 17 F(C₁₀F)₉ Staining of Cellular Material in Cell HealthEvaluation

Lymphoid cancer cell lines were prepared in complete growth medium or byisolation from primary tissue and/or blood samples by density gradients,magnetic cell sorting, or FACS. Cells were then induced, in vitro, intonecrosis and apoptosis to form dead cells, cell debris, and apoptoticbodies by heat or metabolic stress (see Example 14 for stressparameters). Fresh cells were maintained by no inducement.

Cells were then centrifuged and washed using standard flow cytometrywash techniques or washed with staining buffers containing protein,phosphate buffered saline, and metabolic inhibitors. F(C₁₀F)₉ dyesolution was then incubated at 50 M with 0.5 mL of cells at a celldensity of 2-3×10⁶ cells/mL per test well of stain buffer. The mixtureswere incubated at 20-25° C. for 30-40 minutes. After incubation, cellswere washed in 600-800 μL of wash buffer (described in Example 16) andcentrifuged (g×350) for 5 minutes.

DNA intercalating agents 7-aminoactinomycin D (7-AAD) viability stainand propidium iodide (PO-PRO®-1) were used together to identify necroticand apoptotic cells in a multi-color, 5 parameter assay. Cells werecentrifuged and washed to remove excess dye using standard flowcytometry wash techniques or washed with staining buffers containingprotein, phosphate buffered saline, and metabolic inhibitors.

Cells were acquired by flow cytometry and analyzed to identify viablecells, early apoptotic, and late apoptotic bodies present in the cellpreparation. This was achieved by targeting 5000 intact viable cells andkeeping FSC-Lin threshold low enough to measure subcellular debris.Fluorescence detection was performed at 488 nM blue laser line by flowcytometry with peak emission (521 nM) detected using 525/50 bandpassfilter.

When compared to fresh cultures, the staining patterns of this cellpreparation indicate a broad range of fluorescence, and a sensitivity ofdetection in a fresh culture that is not detected with 7-AAD.Fluorescent patterns found in FIG. 11 (i.e. using F(C₁₀F)₉SH) provide ahigh resolution and broad dynamic range of three and a half logfluorescence correlated to cellular structures similar to nucleic acidsas co-stained by 7-AAD and propidium iodide. Detailed information aboutcell vitality can be extracted when dye (i.e. F(C₁₀F)₉ or F(C₁₀F)₉SH) isused alone and when it is combined with other reagents (i.e. 7-AAD andpropidium iodide).

Using this preparation, 4 populations are revealed (live cells [L],apoptotic cells that are not dye positive [A1], cells with low-to-midbrightness [A2], and necrotic cells [D]; see FIG. 12). The fluorescenceintensities reveal a gradient that should correlate to graded states ofmembrane permeability. When intact cells are excluded from the analyses,a detailed pattern of additional live cells/compromised cells emergesthat do not fall within an “intact” cell gated by morphology.

FIG. 13 shows fresh culture and sensitive detection of vitality statesbetween two different cultures and two different morphologyinterpretations of the collected events.

Example 18 Staining of Cells with F(C₁₀F)₉

F(C₁₀F)₉ bulk dye used in the preparation described in Example 17unexpectedly and advantageously stains dead cells much like the aminereactive dye described in Example 16. This indicates that F(C₁₀F)₉ dyein solution does not need to be functionalized in order to bind. Theimpact of this discovery on typical live/dead cell protocol woulddispense the requirement for a protein free incubation buffer anddramatically increase the ease of use for staining dead cells.Traditional amine reactive (i.e. fixable) dyes require a resuspension ofcells in a protein free buffer prior to staining.

Example 19 Patterns and Effects of Dead/Apoptotic Cells

Patterns of mitosis were found in analyzing dead cells. Flow cytometrydata obtained using F(C₁₀F)₉ show patterns that resemble a DNA histogram(see FIGS. 14a-b ). In addition, F(C₁₀F)₉ dye stained cells show apattern that resembles a proliferation arc (see FIGS. 15a-b ). Thesefindings indicate the dyes bind multiple sizes of cellular material thatis not an intact cell but correlate to classical interpretations ofnucleic acid staining patterns of ploidy. Despite the fact that the dyeappears to label “non-intact cells” they are predicted to bind DNA orproteins associated with DNA in fixed and permeabilized cells.

Dye staining shows Jurkat cells that express multiple chromosomes andare in a hyper-diploid state (FIGS. 14a-b ). Dye stained Jurkat andRamos cellular material correlates to classical patterns ofproliferation (FIGS. 15a-b ).

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification, includingU.S. provisional patent application Ser. No. 62/159,771, filed May 11,2015, are incorporated herein by reference, in their entirety to theextent not inconsistent with the present description.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. A compound having the following structure

or a stereoisomer, salt or tautomer thereof, wherein: M is, at eachoccurrence, independently a fluorescent or colored moiety comprisingthree or more aryl or heteroaryl rings, or combinations thereof, L¹, L²and L³ are, at each occurrence, independently an optional alkylene,alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker; L⁴ is, at each occurrence,independently an alkylene, alkenylene, alkynylene, heteroalkylene,heteroalkenylene or heteroalkynylene linker; R¹ is, at each occurrence,independently H, alkyl or alkoxy; R² and R³ are each independently H,OH, SH, alkyl, alkoxy, alkylether, —OP(═R_(a))(R_(b))R_(c), Q, a linkercomprising a covalent bond to Q, a linker comprising a covalent bond toan analyte molecule, a linker comprising a covalent bond to a solidsupport or a linker comprising a covalent bond to a further compound ofstructure (I), wherein: R_(a) is O or S; R_(b) is OH, SH, O, S, OR^(d)or SR^(d); R_(c) is OH, SH, O, S, OR^(d), SR^(d), alkyl, alkoxy,alkylether, alkoxyalkylether, phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether; R⁴ is, ateach occurrence, independently OH, SH, O, S, OR^(d) or SR^(d); R⁵ is, ateach occurrence, independently oxo, thioxo or absent; Q is, at eachoccurrence, independently a moiety comprising a reactive group capableof forming a covalent bond with an analyte molecule, a solid support ora complementary reactive group Q′; R^(d) is a cation; m is, at eachoccurrence, independently an integer of zero or greater, provided thatat least one occurrence of m is an integer of three or greater; and n isan integer of one or greater.
 2. The compound of claim 1, wherein thecompound has the following structure (IA):

wherein x¹, x² and x³ are, at each occurrence, independently an integerfrom 0 to
 6. 3. The compound of claim 1, wherein the compound has thefollowing structure (IB):

wherein: x¹,x² and x³ are, at each occurrence, independently an integerfrom 0 to 6; and y is an integer from 1 to
 6. 4. The compound of claim1, wherein the compound has one of the following structures (IB′) or(IB″):


5. The compound of claim 1, wherein R² and R³ are each independently OHor —OP(═R_(a))(R_(b))R_(c).
 6. The compound of claim 1, wherein one ofR² or R³ is OH or —OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ isQ or a linker comprising a covalent bond to Q.
 7. The compound of claim1, wherein Q comprises a sulfhydryl, disulfide, activated ester,isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide,sulfonyl halide, phosphine, α-haloamide, biotin, amino or a maleimidefunctional group.
 8. The compound of claim 1, wherein Q comprises amoiety selected from Table
 1. 9. The compound of claim 1, wherein one ofR² or R³ is OH or —OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ isa linker comprising a covalent bond to an analyte molecule or a linkercomprising a covalent bond to a solid support.
 10. The compound of claim9, wherein the analyte molecule is an enzyme, receptor, receptor ligand,antibody, glycoprotein, aptamer or prion.
 11. The compound of claim 1,wherein m is, at each occurrence, independently an integer from 3 to 10.12. The compound of claim 1, wherein m is, at each occurrence,independently an integer from 7 to
 9. 13. The compound of claim 1,wherein M is, at each occurrence, independently fluorescent or colored.14. The compound of claim 1, wherein M is, at each occurrence,independently a dimethylaminostilbene, quinacridone,fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine,terrylene, sexiphenyl, porphyrin, benzopyrene,(fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl,(bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl,bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl,squaraine, squarylium, 9,10-ethynylanthracene, ter-naphthyl,p-terphenyl, perylene, azobenzene, phenazine, phenanthroline,thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide,perylene amide, coumarin dye, resorufin dye, dipyrrometheneborondifluoride dye, ruthenium bipyridyl dye, energy transfer dye, thiazoleorange dye, polymethine or N-aryl-1,8-naphthalimide dye moiety.
 15. Thecompound of claim 1, wherein M is, at each occurrence, independentlypyrene, perylene, perylene monoimide or 6-FAM or derivative thereof. 16.The compound of claim 1, wherein M, at each occurrence, independentlyhas one of the following structures:


17. The compound of claim 1, wherein the compound is selected from oneof the following structures:

wherein F, E and Y have the following structures:


18. A method of staining a sample, comprising adding to said sample thecompound of claim 1 in an amount sufficient to produce an opticalresponse when said sample is illuminated at an appropriate wavelength.19. A method for visually detecting an analyte molecule, the methodcomprising: (a) providing the compound of claim 1, wherein R² or R³ is alinker comprising a covalent bond to the analyte molecule; and (b)detecting the compound by its visible properties.
 20. A method forvisually detecting an analyte molecule, the method comprising: (a)admixing the compound of claim 1, wherein R² or R³ is Q or a linkercomprising a covalent bond to Q, with the analyte molecule; (b) forminga conjugate of the compound and the analyte molecule; and (c) detectingthe conjugate by its visible properties.
 21. A composition comprisingthe compound of claim 1 and one or more analyte molecules.
 22. A methodfor determining the presence of dead cells in a sample, the methodcomprising contacting the sample with a compound of claim 1, therebybinding or associating the compound with the dead cells, and observing afluorescent signal from the compound bound or associated with the deadcells.